Human interferon alpha derivative and polyethylene glycol modified substance thereof

A technology of PEGylation and interferon α, which is applied in the field of biomedicine, can solve the problems affecting the purity of PEGylation modifications and the low rate of PEGylation modification, and achieve good clinical application prospects, good water solubility, The effect of low antigenicity

Active Publication Date: 2009-08-26
BEIJING SIHUAN PHARMA +1
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AI Technical Summary

Problems solved by technology

[0008] Sequence analysis found that the first N-terminus of human interferon α family α1a, α2a and α1b is a cysteine ​​residue, and a disulfide bond is formed between -SH and -SH of Cys 29, so that human interferon The polypeptide molecule of α becomes a coiled spatial configuration. If the amino-termin

Method used

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  • Human interferon alpha derivative and polyethylene glycol modified substance thereof
  • Human interferon alpha derivative and polyethylene glycol modified substance thereof
  • Human interferon alpha derivative and polyethylene glycol modified substance thereof

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0034] Example 1: gly-gly-gly-gly-gly-IFNα-2a (hereinafter referred to as Gly(5)-IFN

[0035] α) Secreted expression in Pichia pastoris

[0036] 1. Design and acquisition of target gene:

[0037] The amino acid sequence of IFNα-2a is shown in SEQ ID No. 1. After obtaining the cDNA of IFNα-2a through Genebank, change the corresponding codon to yeast preference, and add the corresponding nucleotide sequence of gly-gly-gly-gly-gly at the N-terminal. This cDNA sequence was used for the construction of the pGAPZα expression plasmid (purchased from invitrogen) of Pichia pastoris GS115 (purchased from invitrogen). Secreted expression is achieved after transformation of GS115 host bacteria. Therefore, the KEX2 enzyme recognition site CTC GAG AAA AGA was added to the design, where CTC GAG is the XhoI restriction site. At the same time, a double stop codon TGA TAA and NotI digestion sequence GCG GCCGC was introduced at the 3'end. The cDNA sequence of IFN used in the yeast expression vector ...

Example Embodiment

[0054] Example 2: Purification of Gly(5)-IFNα

[0055] Step 1: Cationic gel column (CM Sepharose F.F. gel purchased from Amersham Biosciences) chromatography:

[0056] Use pH3.8-4.6 acetate buffer for column loading, elution, and electrophoresis monitoring to collect the target. Then, the target was dialyzed with a pH 7.5-8.5 Tris-HCl buffer solution.

[0057] Step 2: Anion gel column (DEAE Sepharose F.F. gel purchased from Amersham Biosciences) chromatography:

[0058] Use pH 7.5-8.5 Tris-HCl buffer solution for column loading, elution, and collection of the target substance. Then dialyze the target with a pH 7.5-8.5 phosphate buffer solution.

Example Embodiment

[0059] Example 3: Preparation and purification of PEG coupling modified samples

[0060] 1. The Gly(5)-IFNα sample obtained in Example 1 was dialyzed with phosphate buffer (pH 6.0), and then ALD-PEG 40KD (purchased from Beijing Jiankai Technology Co., Ltd.) with a molar ratio of 1:3 was added. Modification is carried out at 2-15°C, and the reaction time is 40 hours. The modified samples obtained were tested by SDS-PAGE. The test results showed that after PEG coupling modification, the molecular weight increased from 19,000 Daltons to nearly 90,000 Daltons, and the modification rate reached about 60%. The target product was obtained. mPEG-Gly(5)-IFNα.

[0061] 2. Purification of mPEG-Gly(5)-IFNα:

[0062] The mPEG-Gly(5)-IFNα was adjusted with acetate buffer (pH value 4.0-5.0) to terminate the reaction, and then purified on a cationic gel column SP Sepharose F.F. gel column. Use NaCl (0.15M) solution for elution, collect the target, and the purity is more than 95%. The result is im...

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Abstract

The invention discloses human interferon alpha derivative and polyethylene glycol modified substance thereof. The human interferon alpha derivative provided by the present invention is formed by connecting 4-7 glycines on amino end of human interferon alpha. The polyethylene glycol modified substance of the human interferon alpha derivative is formed by connecting polyethylene glycol to the amino end of the human interferon alpha derivative through amido bond. The invention also provides a preparation method for the polyethylene glycol modified substance of the human interferon alpha derivative and use thereof in treating and preventing viral infection or tumor medicament. The polyethylene glycol modified substance of the human interferon alpha derivative does not contain non-amino end modified isomer, and has purity of more than 95%, long in vivo retention time, and widely clinic application prospect.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to human interferon alpha derivatives, pegylated modified products of human interferon alpha derivatives and their preparation and application. Background technique [0002] Human interferon (interferon, IFN) is a kind of active protein with broad-spectrum anti-virus, anti-proliferation and immune regulation functions in the body, and it is one of the earliest cytokines used in clinical treatment. Human interferon can be divided into three types, namely human interferon-α, human interferon-β, and human interferon-γ, and can be further divided into several subtypes according to the amino acid sequence of each type of IFN. A large number of clinical studies have proved that human interferon-α is an important anti-tumor and anti-viral drug. At present, the most widely used clinically in my country is mainly recombinant human interferon α-1a, α-2a and α-1b. [0003] Human interfer...

Claims

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Application Information

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IPC IPC(8): C07K14/56C12N15/21C12N15/63C07K17/08A61K38/21A61K47/48A61P31/12A61P35/00A61K47/64
Inventor 夏中宁吴然张丽杰
Owner BEIJING SIHUAN PHARMA
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